Cerium-oxide nanoparticle based device for the detection of reactive oxygen species and monitoring of chronic inflammation

ABSTRACT

A polymer-coated cerium oxide based device and system is disclosed for detecting reactive oxygen species and monitoring chronic inflammation. The device and system encapsulate free therapeutic nanoparticle elements not present in a living body in a prosthetic or implantable unit. Embodiment one is a two-chamber structure with a reactive oxygen species (ROS) scavenging component on one end and at the opposite end is an imaging agent consisting of at least one of a fluorophore capable of fluorescence emission, a chemiluminescent agent, a magnetic relaxation agent and an X-ray contrast agent. Embodiment two is a single chamber device consisting of a multifunctional nanocomposite with a ROS-scavenging nanoparticle constituent (nanoceria) and a multimodal reporting nanoparticle component (i.e. Dex-IO-DiR). The device and system are utilized in treatment of diseases with a pro-inflammatory component, including, but not limited to, Crohn&#39;s disease, ulcerative colitis, inflammatory bowel disease, cystic fibrosis, arthritis, and cancer chemotherapy.

GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Agency contractnumber K01 CA101781 and R01 GM084331 awarded by the National Institutesof Health. The government has certain rights in this invention.

RELATED APPLICATIONS

This invention claims the benefit of priority from U.S. Utility patentapplication Ser. No. 12/924,976 filed Oct. 8, 2010 which claims thebenefit of priority to U.S. Provisional Patent Application Ser. No.61/250,750 filed Oct. 12, 2009. The entire disclosure of each of theapplications listed in this paragraph are incorporated herein byspecific reference thereto.

FIELD OF THE INVENTION

This invention relates to uses of nanoceria particles, and in particularto nanoceria-based methods, systems aid devices useful in detectingreactive oxygen species and monitoring chronic inflammation in vivo.

BACKGROUND AND PRIOR ART

Most recently, it has been found that nanosized cerium oxide (nanoceria)possesses antioxidant activity at physiological pH and has potential usein biomedical applications, such as protection against radiation damage,oxidative stress and inflammation, as reported by various researchers,including R. W. Tarnuzzer, et al. in Nano Lett 2005, 5, 2573; J. P.Chen, et al. in Nature Nanotechnology 2006, 1, 142; J. Niu, et al inCardiovasc Res 2007, 73, 549; M. Das, et al. in Biomaterials 2007, 28,1918 and J. M. Perez, et al. in Small 2008.

Biological systems have evolved to utilize readily available elements,such as carbon, oxygen, hydrogen, nitrogen, calcium and iron. Lessabundant elements may be utilized as co-factors in enzymatic complexfactors, or can be found in chelated forms surrounded by aromatic ringsand coordinated bonding. However, many elements, including rare earths,such as cerium, are not present in living organisms, due to their highlyreactive nature resulting in toxicity.

Hence, cells do not have protective apparatuses against elements notpresent in living organisms, and organisms do not have mechanisms tohandle the storage, utilization and release of these elements from thebody. For instance, lead and arsenic accumulate in the body and can leadto severe pathological conditions, including organ failure and death.Also, nanoparticles composed of metals such as cadmium in Quantum dots(QDOTS) are partially toxic.

Similarly, cerium oxide nanoparticles, despite being potent reactiveoxygen species (ROS) scavengers and selective cytoprotective agents,have shown cellular uptake and intracellular residency of cerium oxidenanoparticles which induces dephosphorylation of various substrates,causing aberrant cell signaling and alterations in the transcriptionaland post-translational levels, as reported by J. M. Perez et al. in“Synthesis of biocompatible dextran-coated nanoceria with pH-dependentantioxidant properties” Small, 2008. 4(5): 552-556, Tan, F., et al. in“An efficient method for dephosphorylation of phosphopeptides by ceriumoxide” J Mass Spectrom, 2008. 43(5): 628-632 and Xia, T., et al. in“Comparison of the mechanism of toxicity of zinc oxide and cerium oxidenanoparticles based on dissolution and oxidative stress properties” ACSNano, 2008. 2(10): 2121-2134.

Furthermore, Asati, A., et al. in “Oxidase-Like Activity ofPolymer-Coated Cerium Oxide Nanoparticles” Angew Chem Int Ed Engl, 2009reported the oxidase-like activity of these nanoparticles in acidicmicroenvironments, which may facilitate the oxidation of intracellularand extracellular components. Most importantly, as cerium is not foundin the human body and there are no clearance mechanisms for it, ceriummay cause toxicity, contrary to iron oxide nanoparticles, where theiron-containing core can be metabolized and uptaken by ferritin andtransferrin, as reported by Xia, T., et al. in “Comparison of themechanism of toxicity of zinc oxide and cerium oxide nanoparticles basedon dissolution and oxidative stress properties” ACS Nano, 2008, supraand Rzigalinski, B. A., “Nanoparticles and cell longevity” TechnolCancer Res Treat, 2005 4(6): 651-659.

In view of the above findings, potential in vivo application of thesenanoparticles has been limited. The prior art in the applications ofnanoceria pertains to the administration and use of the nanoparticlesfree in solution, hence nanoparticle clearance and toxicity may beobserved. No prior art reports the encasing of these nanoparticles in adevice, such as described in the present invention, thereby preventingdirect exposure of the living organism to these nanoparticles.

Thus, a device encapsulating therapeutic elements not present in aliving body, such as cerium oxide, is an attractive alternative,preventing the adverse side effects from free nanoparticles incirculation. Therefore, for these reasons, it would be advantageous tointroduce nanoparticles within devices to minimize their exposure,accumulation and potential toxicity to biological systems. Such anembodiment would be classified as a prosthetic (implantable) device andnot a drug, expediting the adoption of these nanoparticles in therapyand the clinic.

It is desirable to extend the utility of the coated nanoceria particlesas a stable, effective, therapeutic device, system or method fordetecting reactive oxygen species and monitoring chronic inflammation inbiological tissue, while causing no adverse side effects or toxicity tothe body. The present invention provides a much needed weapon in thearsenal for treating a broad range of ailments with a pro-inflammatorycomponent, for cancer therapy, for patients with transplants orprosthetic devices and the like.

SUMMARY OF THE INVENTION

A primary objective of the present invention is to provide a device,system and method encapsulating therapeutic elements not present in aliving body, such as cerium oxide nanoparticles, in a prosthetic orimplantable unit thereby preventing adverse side effects from freenanoparticles in circulation.

A secondary objective of the present invention is to provide a ceriumoxide nanoparticle-based device, system and method for the detection ofreactive oxygen species (ROS).

A third objective of the present invention is to provide a cerium oxidenanoparticle-based system, method and device for monitoring chronicinflammation.

A fourth objective of the present invention is to provide a cerium oxidenanoparticle-based system, method and device for the detection ofreactive oxygen species (ROS) and monitoring of chronic inflammation,through the co-encasing of cerium oxide and fluorescent polymeric ironoxide nanoparticles (including Dex-IO-DiR and PAA-IO-DiI) in atwo-chamber biocompatible device utilizing the potent ROS scavengingactivity of cerium oxide nanoparticles.

A fifth objective of the present invention is to provide a cerium oxidenanoparticle-based system, method and device for the detection ofreactive oxygen species (ROS) and monitoring of chronic inflammation,through the interspersing of cerium oxide and fluorescent polymeric ironoxide nanoparticles (including Dex-IO-DiR and PAA-IO-DiI) in a singlechamber biocompatible device utilizing the potent ROS scavengingactivity of cerium oxide nanoparticles.

A sixth objective of the present invention is to provide a stable,effective, cerium oxide nanoparticle-based therapeutic device, systemand method for detecting reactive oxygen species and monitoring chronicinflammation in biological tissue, while causing no adverse side effectsor toxicity to the body.

A seventh objective of the present invention is to provide monitoring ofinflammation levels by changes in fluorescence emission (increase ordecrease in the signal measured via optical methods), changes inmagnetic relaxation (increase or decrease in the signal assessed viaMRI), or alterations in other modalities' properties such as, X-raycontrast).

A preferred device of the present invention encapsulates freetherapeutic nanoparticle elements not present in a living body in aprosthetic or implantable unit having a first chamber for a reactiveoxygen species (ROS) scavenging component having an exteriorsemi-permeable membrane surface and an interior cavity containing theROS scavenging component, a second chamber for an imaging agent havingan exterior semi-permeable membrane surface and interior cavitycontaining the imaging agent, and the first chamber and the secondchamber joined in a non-communicating manner so that both the firstchamber and the second chamber independently communicate with an aqueousenvironment through the exterior semi-permeable membrane of each chamberallowing the diffusion of ROS from the environment on the exterior ofthe first chamber and the exterior of the second chamber such that thedevice can sense elevated non-physiological reactive oxygen species(ROS) levels associated with at least one of the following conditions,inflammation, exposure to radiation, and changes in a tissue/cellularmicro-environment.

In the preferred device, the ROS-scavenging component in the firstchamber is a plurality of nanoparticles selected from at least one of apolymer-coated cerium oxide that is coated with at least one of dextranand polyacrylic acid, and polymeric nanoparticles encapsulating a “soft”ROS-scavenging agent, selected from at least one of polymericnanoparticles encapsulating proteins, polymeric nanoparticlesencapsulating chemicals and polymeric nanoparticles encapsulating asmall molecule.

It is preferred that the protein encapsulated by polymeric nanoparticlesis selected from at least one of catalase, superoxide dismutase and thechemical encapsulated by polymeric nanoparticles is peroxidase and thesmall molecule encapsulated by polymeric nanoparticles is reducedglutathione.

In the preferred device, the second chamber contains an imaging agentselected from at least one of a fluorophore capable of fluorescenceemission, a chemiluminescent agent, a magnetic relaxation agent and anX-ray contrast agent encapsulated within a plurality of polymericcavities in a polymer-coated nanoparticle selected from at least one ofiron oxide, bismuth, europium, gadolinium and chelates thereof, coatedwith dextran, PAA, HBPE, PLGA, and mixtures thereof.

The preferred imaging agent is a hydrophobic fluorophore selected fromat least one of octodecyl indocarbocyanine (DiI), indodicarbocyanine(DiD) and indotricarbocyanine (DiR) encapsulated within the plurality ofpolymeric cavities of the polymer-coated nanoparticle selected from atleast one of iron oxide, bismuth, europium, gadolinium and chelatesthereof, coated with dextran, PAA, HBPE, PLGA, and mixtures thereof.

It is also preferred that the imaging agent in the second chamber is ahydrophilic fluorophore selected from at least one of cyanine 5.5(Cy5.5) and ICG encapsulated within the plurality of polymeric cavitiesof the polymer-coated nanoparticle of iron oxide, bismuth, europium,gadolinium and chelates thereof, coated with dextran, PAA, HBPE, PLGA,and mixtures thereof.

It is further preferred that the polymer-coated nanoparticles of ironoxide are coated with polymers selected from dextran and polyacrylicacid that are used to monitor reactive oxygen species generation throughmagnetic resonance imaging (MRI). The polymer-coated nanoparticles ofiron oxide are also used to monitor inflammation and upregulatedreactive oxygen species (ROS) production through fluorescence-basedoptical methods.

The reactive oxygen species (ROS) scavenging component of the preferreddevice protects the imaging agent from exposure to ultraviolet (UV)radiation.

A second embodiment of a preferred device encapsulates free therapeuticnanoparticle elements not present in a living body in a prosthetic orimplantable unit having a single chamber containing a reactive oxygenspecies (ROS) scavenging component interspersed with an imaging agent ina preselected ratio of 1:1, 1:2 and 2:1, whereby the scavengingcomponent and the imaging component simultaneously communicate with anaqueous environment through the exterior semi-permeable membrane of thesingle chamber allowing the diffusion of ROS from the environment on theexterior of the chamber such that the device can sense elevatednon-physiological reactive oxygen species (ROS) levels associated withat least one of the following conditions, inflammation, exposure toradiation and changes in a cellular micro-environment; thereby forming asingle chamber device consisting of a nanocomposite, derived from theconjugation and linking of polymer-coated nanoceria with at least one ofthe following nanoparticles, Dex-IO-DiR, PAA-IO-DiR and mixturesthereof.

It is preferred that the ROS scavenging component in the single chamberis a plurality of nanoparticles selected from a polymer-coated ceriumoxide coated with at least one of dextran and polyacrylic acid, andpolymeric nanoparticles encapsulating a “soft” ROS-scavenging agent,selected from at least one of polymeric nanoparticles encapsulatingproteins, polymeric nanoparticles encapsulating chemicals and polymericnanoparticles encapsulating a small molecule.

It is also preferred that the protein encapsulated by polymericnanoparticles is selected from at least one of catalase, superoxidedismutase and the chemical encapsulated by polymeric nanoparticles isperoxidase and the small molecule encapsulated by polymericnanoparticles is reduced glutathione.

The preferred imaging agent in the single chamber is selected from afluorophore capable of fluorescence emission, a chemiluminescent agent,a magnetic relaxation agent and an X-ray contrast agent encapsulatedwithin a plurality of polymeric cavities in a polymer-coatednanoparticle selected from at least one of iron oxide, bismuth,gadolinium, chelates of gadolinium and europium.

The imaging agent is a hydrophobic fluorophore selected from at leastone of octodecyl indocarbocyanine (DiI), indodicarbocyanine (DiD) andindotricarbocyanine (DiR) encapsulated within the plurality of polymericcavities of the polymer-coated nanoparticle selected from at least oneof iron oxide, bismuth, gadolinium, chelates of gadolinium and europium.

It is most preferred that the imaging agent in the single chamber is ahydrophilic fluorophore selected from at least one of cyanine 5.5(Cy5.5) and indocyanine green (ICG) encapsulated within the plurality ofpolymeric cavities of the polymer-coated nanoparticle of iron oxide,europium, bismuth, gadolinium, chelates of gadolinium, and mixturesthereof.

The polymer-coated nanoparticles of iron oxide are coated with polymersselected from dextran and polyacrylic acid are used to monitor reactiveoxygen species generation through magnetic resonance imaging (MRI) andare also used to monitor inflammation and upregulated reactive oxygenspecies (ROS) production through fluorescence-based optical methods.

The reactive oxygen species (ROS) scavenging component of the singlechamber device protects the imaging agent from exposure to ultraviolet(UV) radiation.

A preferred nanocomposite system encapsulating free therapeuticnanoparticle elements not present in a living body requires thepreparation of a miniature device containing a reactive oxygen species(ROS) scavenging agent and a fluorophore imaging agent, implanting theminiature device in a physiological environment, exposing the miniaturedevice to non-physiological ROS levels, and monitoring the near-infraredfluorophore changes and magnetic relaxation time shifts, signaling theabnormal ROS concentration.

The preferred nanocomposite system further consisting of a miniaturedevice, that is exposed to elevated ROS levels and prevents theregeneration of the ROS scavenger, leading to multimodal sensing changesin fluorescence emission and an increase in the magnetic relaxation(MRI) times of the nanocomposite.

The preferred nanocomposite system further comprising multimodaltracking, having as components nanoparticles with chemiluminescence,magnetic relaxation or X-ray contrast agents.

The preferred nanocomposite system is transplantable as a distinctentity or part of a prosthetic device and is utilized in diseases with apro-inflammatory component selected from the group consisting of,Crohn's disease, ulcerative colitis, inflammatory bowel disease, cysticfibrosis, and arthritis.

The nanocomposite system has a miniature device that is also utilized ina treatment regime monitoring the potential induction of inflammation inat least one of cancer chemotherapy and antimicrobial therapy and alsoin real-time monitoring of ROS during the synthesis of ROS-sensitivecompounds.

The preferred nanocomposite system is also used in tissue protectionagainst elevated ROS levels, in chemotherapy, prosthesis, andpost-transplant operations.

Further objects and advantages of the present invention will be apparentfrom the following detailed description of a presently preferredembodiment, which is illustrated schematically in the accompanyingdrawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a conceptual diagram of the in vivo inflammation monitoringdevice of the present invention having two non-communicating chambers.

FIG. 2 is a schematic representation of the operation principle of thein vivo inflammation monitoring device of the present invention.

FIG. 3 is a graph of the time-dependent fluorescence of thenanoparticles (HBPE-DiI) in the presence of reactive oxygen species(ROS).

FIG. 4 is a graph showing the decrease in the fluorescence emission ofnanoparticles after 24 hours in the presence of hydrogen peroxide(H₂O₂).

FIG. 5 is a graph of time-dependent nanoparticle fluorescence emissionin the presence of nanoceria and ROS in suspension.

FIG. 6 is a graph of fluorescence intensity of nanoceria particles inwater and in H₂O₂ showing that nanoceria provides sustained protectionfrom ROS to fluorescent nanoparticles.

FIG. 7 is a graph of time-dependent retention of the fluorescenceemission of fluorescent nanoparticles (HBPE-DiI) by nanoceria afterexposure to ultraviolet (UV) radiation.

FIG. 8A shows a cerium oxide nanoparticle-based device wherein thefluorescence emission of the DiI-Dex-IO nanoparticles is stable underphysiological conditions (no ROS i.e., no H₂O₂, pH 7) as determinedafter 24 hour incubation.

FIG. 8B shows a cerium oxide nanoparticle-based device wherein thefluorescence emission of the DiI-Dex-IO nanoparticles is diminishedafter 24-hour-long exposure to ROS(H₂O₂, pH 7).

FIG. 8C shows that a cerium oxide nanoparticle-based device withpolymer-coated, polyacrylic acid (PAA), nanoceria does not affect thefluorescence emission of the DiI-Dex-IO nanoparticles after 24-hour-longincubation at physiological conditions (no ROS i.e., no H₂O₂, pH 7)

FIG. 8D shows that a cerium oxide nanoparticle-based device withpolymer-coated, polyacrylic acid (PAA), nanoceria does not affect thefluorescence emission of the DiI-Dex-IO nanoparticles from ROS after24-hour exposure to H₂O₂ at pH 7.

FIGS. 9A, 9B, 9C shows that a cerium oxide nanoparticle-based devicewith polymer-coated, polyacrylic acid (PAA), nanoceria protects thefluorescence emission of the DiI-Dex-IO nanoparticles from ROS for aperiod of 24 hours at physiological H₂O₂ concentrations of 0.5 μM, 1.5μM and 3.0 μM in solutions with a pH value of 7.

FIG. 10A shows that a cerium oxide nanoparticle-based device withpolymer-coated, polyacrylic acid (PAA), nanoceria does not protect thefluorescence emission of the DiI-Dex-IO nanoparticles from ROS for aperiod of 24 hours at a non-physiological pH value of 4 in the presenceof H₂O₂, allowing imaging of ROS generation.

FIG. 10B shows that a cerium oxide nanoparticle-based device withpolymer-coated, polyacrylic acid (PAA), nanoceria does not protect thefluorescence emission of the DiI-Dex-IO nanoparticles from ROS for aperiod of 24 hours during inflammatory conditions and the presence ofH₂O₂, allowing imaging of ROS generation.

FIG. 11 is a graph showing the percent change in fluorescent intensityof a cerium oxide nanoparticle-based device with polymer-coated,polyacrylic acid (PAA), nanoceria wherein the fluorescence emission ofthe DiI-Dex-IO nanoparticles changes with H₂O₂ concentrations of 0.5 μM,1.5 μM, 3.0 μM, 1.0 μM at pH value 4 and 6.0 μM.

FIG. 12 is a graph of the stability of the non-encapsulated fluorophore(DiI) at physiological and acidic pH values, pH 7.5 and pH 4,respectively.

FIG. 13 is a graph of the fluorescence emission stability of thenon-encapsulated fluorophore (DiI) at physiological and acidic pHvalues, pH 7.5 and pH 4, respectively

FIG. 14 is a chart showing the regeneration capacity of polymer-coated,polyacrylic acid (PAA), nanoceria at pH 7; at pH 7 and 1.0 μM H₂O₂; atpH 4 and 1.0 μM H₂O₂; and at pH 7 and 6.0 μM H₂O₂; at low pH or highinflammation the nanoceria component cannot regenerate after exposure toreactive oxygen species (ROS).

FIG. 15 is a schematic depiction of the detection of ROS with a deviceconsisting of a nanoceria ROS-scavenging chamber and a multimodalDiR-Dex-IO nanoparticle (NIR-fluorescent/MRI) monitoring chamber.

FIG. 16A shows that a cerium oxide nanoparticle-based device with nopolyacrylic acid (PAA) coated nanoceria and with multimodal DiR-Dex-IOnanoparticles is sensitive towards ROS generated by H₂O₂ (1.0 μM), after24-hour exposure to H₂O₂ in pH 7 (physiological pH). The ROS beacons aremultimodal and can sense ROS via changes in fluorescence and T2relaxation times (T2_(in)=165 ms, T2_(fin)=280 ms).

FIG. 16B shows that a cerium oxide nanoparticle-based device with bothpolymer-coated, polyacrylic acid (PAA) nanoceria and multimodalDiR-Dex-IO nanoparticles can differentiate physiological ROS levelsgenerated by H₂O₂ (1 μM), after 24-hour exposure to H₂O₂ in pH 7(physiological pH). At physiological pH and ROS levels, the ROS beaconsretain their fluorescence emission intensity and a small change in theT2 relaxation times are observed (T2_(in)=165 ms, T2_(fin)=220 ms).

FIG. 17A shows the clustering that is observed in the multimodalDiR-Dex-IO nanoparticles upon incubation without ROS, i.e., H₂O₂.

FIG. 17B shows the clustering that is observed in the multimodalDiR-Dex-IO nanoparticles upon incubation with ROS, i.e., H₂O₂.

FIG. 18 shows the corresponding change in T2 relaxation signal (MRIsignal) upon addition of H₂O₂.

FIG. 19 is a conceptual diagram of the in vivo inflammation monitoringdevice of the present invention having a single chamber, which alsoshows the cross-sectional view of the device's fluorescence and MRIresponse to physiological and non-physiological conditions.

FIG. 20 is a side-view of the fluorescence image of the single-chamberdevice consisting of polymer-coated, polyacrylic acid (PAA) nanoceriaand DiR-Dex-IO nanoparticles after 24-hour-long incubation atphysiological conditions (1: no ROS i.e. no H₂O₂, pH 7), at aphysiological H₂O₂ concentration (2: 1 μM H₂O₂, pH 7), at anon-physiological inflammatory H₂O₂ concentration (3: 6 μM H₂O₂, pH 7),and at a non-physiological chronic inflammatory H₂O₂ concentration andnon-physiological pH (4: 6 μM H₂O₂, pH 4).

FIG. 21 is a top-view of an MRI image of the single-chamber consistingof polymer-coated, polyacrylic acid (PAA) nanoceria and DiR-Dex-IOnanoparticles after 24-hour-long incubation at physiological (1: no ROSi.e. no H₂O₂, pH 7; 2: 1 μM H₂O₂, pH 7) or non-physiological conditions(3: 6 μM H₂O₂, pH 7; 4: 6 μM H₂O₂, pH 4).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before explaining the disclosed embodiments of the present invention indetail it is to be understood that the invention is not limited in itsapplication to the details of the particular arrangements shown sincethe invention is capable of other embodiments. Also, the terminologyused herein is for the purpose of description and not of limitation.

Chemical names for fluorophores:

DiI—1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate

DiD—1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate

DiR—1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide

ICG—indocyanine green (1H-Benz[e]indolium,2-[7-[1,1-dimethyl-3-(4-sulfobutyl)benz[e]indolin-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulfobutyl)-,hydroxide)

Cy5.5—cyanine 5.5

Dex-IO-DiR is an abbreviation used herein to mean dextran-coated ironoxide with indotricarbocyanine (DiR) encapsulated in the polymericdextran coating.

GSNO is an acronym for N—(N-L-y-glutamyl-5-nitroso-L-cysteinyl)-glycine

HBPE-DiI is an abbreviation used herein to mean hyper-branched polyesternanoparticles encapsulating indocarbocyanine (DiI).

MRI is an acronym for magnetic resonance imaging.

PAA is the acronym used herein to mean polyacrylic acid.

PAA-IO-DiI is an abbreviation used herein to mean polyacrylicacid-coated iron oxide nanoparticles encapsulating indocarbocyanine(DiI).

PLGA is the acronym used herein to mean Poly-Lactic-Co-Glycolic Acid.

“Physiological” is a term used herein to mean a characteristic of orappropriate to an organism's healthy or normal functioning which ismeasured by such factors as pH value, inflammation levels and the like.For example, the normal pH range of the intracellular fluid of cells is6.9-7.4. One of the most important processes in the human body is theprocess by which the pH balance is maintained. Body pH measures thenumber of hydrogen ions in solution within the body. The pH scale rangesfrom 0-14 with <6.4 acidic and >6.4 alkaline for body fluid, notincluding blood which is a normal 7.3 pH. An acidic pH has a low abilityto attract hydrogen ions, while an alkaline solution has a high abilityto attract hydrogen ions. “p” stands for potential and “H” stands forhydrogen; henceforth, the potential of the body to attract hydrogen ionsto secure balance and health.

ROS is an acronym for reactive oxygen species and for the researchpurposes herein hydrogen peroxide (H₂O₂) is used as a representative ROSchemical reagent.

The term “nanoceria” is used interchangeably with “cerium oxidenanoparticles” and is used to refer to the cerium oxide particles ofmultiple valences (Ce⁺³/Ce⁺⁴).

The present invention is capable of several embodiments which include areactive oxygen species (ROS) scavenging agent, an imaging agent andmultiple methods for the conjugation of the scavenging agent and imagingagent in a nanocomposite device, as discussed in more detail below.

ROS Scavenging Agent:

The ROS scavenging agent used in the device consists of polymer-coatedcerium oxide nanoparticles which have high antioxidant/free radicalscavenging activity. The nanoparticles can be prepared using differentsynthetic protocols, including water-based formulations. At normal pH 7nanoceria can effectively sequester ROS generated from various sources,including UV radiation, inflammation and chemical decomposition, asoccurs with H₂O₂ and GSNO(S-nitroso-L-glutathione), due to thereversible switch from Ce⁺³ and Ce⁺⁴ states. This property renderscerium oxide an ideal ROS-scavenging agent. Moreover, atnon-physiological pH or likely under conditions of enhanced ROSgeneration, nanoceria is not able to perform the autocatalyticreversible switching between its two valence states. This allowsnanoceria to serve as a binary switch (ON/OFF) for aberrant ROSproduction. Apart from nanoceria, other ROS scavenging agents can beused, including “soft” ROS scavenging agents such as polymericnanoparticles encapsulating proteins or chemicals, including, but notlimited to, catalase, superoxide dismutase, peroxidase, and glutathione.“Soft” is used herein to mean non-metallic. Examples of “soft” ornon-metallic ROC agents include, but are not limited to, polymericnanoparticles encapsulating proteins, polymeric nanoparticlesencapsulating chemicals such as, catalase, superoxide dismutase,peroxidase and small molecules, such as, reduced glutathione.

Imaging Agent:

The imaging agent consists of multimodal polymer-coated iron oxidenanoparticles, due to the nanoparticles' enhanced magnetic propertiesand FDA approval as clinical imaging agents for MRI. The polymer-coatediron oxide nanoparticles can be synthesized using diverse protocols,including the water-based alkaline precipitation method. Effectivedoping of the nanoparticles with a fluorophore can be achieved viadifferent methodologies, such as the water diffusion procedure or viadirect chemical conjugation. Apart from iron oxide nanoparticles,nanoparticles with gadolinium/gadolinium chelates, bismuth, Qdots, andeuropium can be used. A fluorophore, a conductive polymer or afluorophore-polymer conjugate that is sensitive to ROS can serve as animaging agent of ROS. Furthermore, imaging of ROS can be achieved viachanges in the relaxation times of the nanoparticle solution (in thecase of iron oxide T2).

Multimodal Nanocomposite:

The multifunctional and multimodal nanocomposite can be created by theconjugation of a ROS sensing nanoparticle formulation with an imagingnanoparticle preparation. Efficient nanocomposite formation can beachieved using various chemistries, including carbodiimide for aminatedand carboxylated nanoparticles, or “click” chemistry forazide-propargylated nanoparticles. Hence for instance, the nanocompositecan be obtained by the “click” conjugation of propargylated PAA-coatednanoceria and azide-carrying DiR-PAA-IO, or carbodiimide conjugation ofPAA-coated nanoceria and aminated DiR-Dex-IO. Overall though, eitherconstituent of the nanocomposite can be of “soft” or “hard”nanoparticles. Specifically, “soft” pertains to polymeric nanoparticlesencapsulating a ROS scavenging agent of non-metallic nature (i.e. smallmolecule, protein) or an imaging agent entrapped within polymericmicrodomains and cavities (i.e. fluorophore, chelated ions). On theother hand, “hard” nanoparticles include metallic nanoparticles, such asthe ROS sensing polymer-coated nanoceria, polymer-coated iron oxide, andQuantum dots.

In the present invention, the cerium oxide nanoparticles or nanoceriaare used in a biocompatible device for the detection of reactive oxygenspecies (ROS) and monitoring of chronic inflammation through theco-encasing of cerium oxide and fluorescent polymeric iron oxidenanoparticles, in an exemplary embodiment. Nanoparticle sizes areprovided as an example, not for limitation of the invention. PAA-coatednanoceria can have particles that are approximately 6-8 nanometers (nm)in diameter; Dextran-coated nanoceria has particles that areapproximately 10-14 nm in diameter; PAA-coated iron oxide nanoparticlesare approximately 70-100 nm in diameter; Dextran-coated iron oxidenanoparticles (spherical) are approximately 60-120 nm in diameter; HBPEnanoparticles are approximately 100-130 nm in diameter.

Apart from a near-infrared fluorophore that can achieve deep tissueimaging by circumventing auto-fluoresence in mammalian tissue, ironoxide nanoparticles were used for magnetic resonance imaging (MRI) withbetter spatial resolution and deeper penetration. Results indicate thatthe device exhibits near-infrared fluorescence emission, underphysiological or transient inflammatory conditions with low ROSconcentrations. However, under prolonged exposure to ROS, simulatingchronic inflammation, or in acidic and ROS-rich microenvironmentsfluorescence decreases significantly, due to the catalytic inactivationof cerium oxide nanoparticles and the fluorophore's susceptibility toROS. Furthermore, we show that under elevated ROS, the iron oxidenanoparticles yield high T2 (spin-spin) relaxation times, which can beused as clinically applicable MRI tags for inflammation imaging.

FIG. 1 is a conceptual diagram of an in vivo inflammation monitoringdevice having a first chamber 10 contains antioxidant nanoparticles,such as nanoceria that functions as the reactive oxygenscavenging/sensing (ROS) chamber which is joined to a second chamber 20contains nanoparticles that are fluorescent, magnetic or multimodal andis known herein as the monitoring chamber. Thus, construction of theinflammation monitoring device has two components; a first componentconsisting of a nanoparticle or a nanocomposite with cerium oxide, aROS-sensitive/scavenging probe, and second component consisting ofimaging agents, fabricated through the utilization of material synthesistechniques and conjugation chemistry approaches.

FIG. 2 is a schematic representation of the monitoring device'soperating principle. The device consists of polymer-coated cerium oxidenanoparticles in a first chamber 10 and fluorophore-containingnanoparticles in a second chamber 20. The latter nanoparticles can beeither fluorescent polymeric (i.e. hyperbranched polyester-HBPE-DiI) orpolymer-coated iron oxide nanoparticles (i.e. DiR-Dextran-IO). Each oneof the device's chambers independently communicates with the aqueousenvironment through a semi-permeable membrane, freely allowing thediffusion of ROS from the exterior to the device's chambers. Note thatthere is no communication between the two chambers. No communicationmeans there is not any exchange of particles between the two chambersthat are held in position by an adhesive or glue.

Under conditions of no or mild inflammation a, the device's cerium oxidenanoparticle component (nanoceria) in chamber 11 scavenges the generatedROS, as ROS production is either or transient, leading to preservationof the device's fluorescence emission in chamber 22. However, underconditions of chronic persistent inflammation b, nanoceria in chamber 12does not have the ability to regenerate quickly enough, thus the excessROS dim or switch off the device's fluorescence emission in chamber 24,due to the fluorophore's sensitivity to ROS.

FIG. 3 is a graph of the time-dependent fluorescence of the imagingnanoparticles in the presence of hydrogen peroxide. One hundred percentfluorescence intensity in water decreases to approximately 60%fluorescence intensity in 1400 minutes in the presence of hydrogenperoxide, a generator of reactive oxygen species (ROS). Thephotostability of the nanoparticles in water (control), is shown by thefluorescence intensity remaining constant. In contrast, in the presenceof hydrogen peroxide (H₂O₂ 1 □□), the fluorescence emission of theHBPE-DiI nanoparticles decreases, due to the presence of ROS generatedby H₂O₂ decomposition that oxidizes the dye and therefore reduces itsfluorescence intensity.

FIG. 4 graphically depicts the decrease in the fluorescence emission ofHBPE-DiI nanoparticles after 24 hours in the presence of hydrogenperoxide. Fluorescence emission spectroscopy indicates the stability ofthe fluorophore nanoparticle (HBPE-DiI) in water and in the absence ofROS. At approximately 300 nm in wavelength, fluorescent intensity ismeasured at 1.0×10⁷ cps. The fluorescence emission significantlydecreases under elevated ROS concentrations due to decomposition ofhydrogen peroxide (1 □□) and at approximately 300 nm wavelengthfluorescence intensity is 6.0×10⁶ cps.

FIG. 5 is a graph of the time-dependent HBPE-DiI nanoparticlefluorescence emission in the presence of nanoceria and ROS insuspension. The graph demonstrates that nanoceria facilitates theretention of the device's fluorescence emission (HBPE-DiI) in thepresence of hydrogen peroxide (1 □□), because nanoceria is able toscavenge the generated ROS therefore preserving the fluorescenceemission of the dye. Over a period of approximately 1400 minutes,fluorescence intensity remains at approximately 95% of the initialfluorescence. Thus, nanoceria protects the fluorophore DiI containingcomponent from ROS.

FIG. 6 is a graph showing that nanoceria provides sustained protectionfrom ROS to fluorescent nanoparticles. Fluorescence emissionspectroscopy supports the hypothesis that the nanoceria component of thedevice would protect the fluorescence emission of thefluorophore(DiI)-containing component, even in the presence of hydrogenperoxide (1 □M).

FIG. 7 is a graph of the time-dependent retention of the fluorescenceemission of the HBPE-DiI fluorescent nanoparticle by nanoceria afterexposure to UV radiation. In the absence of nanoceria, thenon-encapsulated fluorophore (DiI) rapidly loses its fluorescenceemission. Encapsulation of the dye (HBPE-DiI) prolongs the fluorescenceemission from the fluorophore, but gradually it decreases. Addition ofnanoceria to the fluorescent nanoparticles protects fluorescenceemission over prolonged exposure to UV radiation. The fluorescenceintensity remains at 100% of the initial intensity for over 160 minutes.

Collectively, FIGS. 3-7 demonstrate three important results of testingthe two-chamber device in solution. First, in the presence of ROS,fluorescence emission from dyes, such as DiI, rapidly decrease makingthem suitable probes for ROS monitoring. Second, the addition ofnanoceria results in photoprotection of the fluorescence intensity ofthe dye, protecting the fluorophore from ROS attacks. Third, nanoceriaprotects the fluorophore from direct damage and ROS generated during UVradiation.

Photographic Evidence of Function of Polyacrylic Acid (PAA) CoatedNanoceria.

The photographs were taken using a small animal imaging station (IVIS,Xenogen). However, these results can be obtained with other optical andfluorescence instrumentation, including other proprietary small animalimaging stations.

FIG. 8A shows that when there is no coating on nanoceria particles andno H₂O₂ is present, the fluorescence emission of the DiI-Dex-IOnanoparticles is constant/unaltered.

In contrast, FIG. 8B shows that a nanoparticle-based device with nocoating on nanoceria and with 1 μM H₂O₂ present in a solution with a pHvalue of 7 exhibits change in the fluorescence emission of itsDiI-Dex-IO nanoparticles after 24-hour exposure to ROS; there is asignificant loss in fluorescence emission from the device.

FIG. 8C shows that polymer-coated nanoceria (e.g., with polyacrylic acid(PAA) coating), does not affect the fluorescence emission of theDiI-Dex-IO nanoparticles. Most importantly as shown in FIG. 8D,polymer-coated nanoceria protects the fluorescence emission of theDiI-Dex-IO nanoparticles from ROS after 24-hour exposure to H₂O₂ at pH7; there is no change in fluorescence emission.

Thus, original photographs of the device (FIGS. 8A, 8C and 8D) show thatnanoceria does not affect DiR-Dex-IO fluorescence emission underphysiological conditions, and the fluorescence of the DiR-Dex-IO isstable over time, either with or without nanoceria. However, ROS due toH₂O₂ does cause a decrease in the fluorescence emission of DiR-Dex-IOwhen nanoceria is not present (FIG. 8B); but in the presence ofnanoceria, fluorescence is protected as nanoceria scavenges ROS at pH 7;the photobleaching effect of hydrogen peroxide is abrogated andfluorescence emission is retained (FIG. 8D). Incubations for 24 hourswere performed to check stability and long-term exposure to ROS.

FIGS. 9A-9C show that a cerium oxide nanoparticle-based device withpolymer coated, polyacrylic acid (PAA), nanoceria protects thefluorescence emission of the DiI-Dex-IO nanoparticles from ROS after24-hour exposure at physiological H₂O₂ concentrations of 0.5 μM (FIG.9A), 1.5 μM (FIG. 9B) and 3.0 μM (FIG. 9C) in solutions with a pH valueof 7. Stated in another way, under physiological ROS levels ([H₂O₂]=0-3□M), nanoceria protects the fluorescence emission of the DiI-Dex-IOnanoparticles of an implantable therapeutic device.

FIG. 10A shows that a cerium oxide nanoparticle-based device withpolymer coated, polyacrylic acid (PAA), nanoceria does not protect thefluorescence emission of the DiI-Dex-IO nanoparticles from ROS after24-hour exposure to H₂O₂ at a non-physiological pH value of 4, allowingimaging of ROS generation.

FIG. 10B shows that a cerium oxide nanoparticle-based device withpolymer coated, polyacrylic acid (PAA), nanoceria does not protect thefluorescence emission of the DiI-Dex-IO nanoparticles from ROS after24-hour exposure under inflammatory conditions simulated by high levelsof ROS, generated by H₂O₂.

Thus, FIGS. 10A and 10B show that under conditions of elevatedinflammation ([H₂O₂]≧6 □□) or nanoceria's pH-dependent catalyticinactivation (acidic pH, i.e., 4.0), the device's fluorescence emissionfrom DiI-Dex-IO nanoparticles decreases. It is worth mentioning that invivo chronic inflammation is often associated with a decrease in the pH,thus it can contribute to decreased fluorescence emission. Hence, thesedata demonstrate that the device's “switch off” occurs specificallyunder non-physiological conditions, such as, high ROS and/or low pH,making the device suitable for in vivo imaging of inflammation.

In other words, during normal levels of inflammation the device will beresponsive and nanoceria in the ROS-scavenging chamber will be able toscavenge ROS, preserving the monitoring chamber's fluorescence emissionherein designated as the ON state. However, during chronic inflammationnanoceria will not be able to scavenge ROS and the device's performancewill be affected, leading to decrease in fluorescence emission hereindesignated as the OFF state. Therefore, the decrease in fluorescenceemission will be an indicator for the physician to take further action,minimizing inflammation and preventing systemic tissue damage.

FIG. 11 is a graph of the change in fluorescence intensity underphysiological and non-physiological conditions. The percentage decreasein the device's fluorescence emission (DiI-Dex-IO) upon polymer-coatednanoceria's ROS-scavenging inactivation was from approximately 100%intensity when H₂O₂ concentrations were 0.5 μM, 1.5 μM and 3.0 μM. Thefluorescence emission intensity decreased to approximately 60% intensitywhen H₂O₂ concentrations were 1.0 μM at pH value 4. Fluoresence emissionintensity decreased to approximately 30% when H₂O₂ concentrations were6.0 μM, at conditions of elevated ROS levels.

FIG. 11 shows that in the presence of nanoceria, physiological ROSlevels do not affect the device's fluorescence. This indicates thespecificity and sensitivity of the device. However, in the presence ofnanoceria, elevated ROS levels associated with aberrant inflammationcause decrease in fluorescence emission (FIG. 11). Thus, the device canbe used for the monitoring of the inflammatory response.

Also, in the presence of nanoceria and low pH, fluorescence emissiondecreases in the presence of ROS (FIG. 11). Hence, the device can beused for the monitoring of ROS generation during chemotherapy orgastrointestinal inflammation, such as ulcerative colitis, inflammatorybowel disease, and Crohn's disease.

Study of Factors that Reduce Fluoresence Emission of Fluorophores

To corroborate that the reduction in the fluorophore emission is due toROS-induced damage and not due to the acidic pH, we performed thefollowing experiments with the fluorophore being in physiological (pH7.5) and acidic (pH 4) levels. No nanoceria was used in these studies inorder to ascribe the role of pH on fluorophore stability.

FIG. 12 shows that the non-encapsulated fluorophore (DiI) has an almostidentical absorbance curve at physiological (pH 7.5) and acidic (pH 4)pH, indicating stability of the fluorophore under both pH values.Ultraviolet (UV)-vis spectroscopy reveals the fluorophore's (DiI)stability at physiological and acidic pH solutions, in the absence ofhydrogen peroxide. Therefore, changes in fluorescence emission are dueto exposure to ROS and not due to the low pH per se.

FIG. 13 is a graph of fluorescence intensity, and shows fluorescenceemission stability of the non-encapsulated fluorophore (DiI) atphysiological (pH 7.5) and acidic (pH 4) values. Fluorescence emissionspectroscopy indicates that the device's fluorophore emission (DiI) ispH-independent. No changes are observed in the emission profile betweenphysiological and acidic pH.

Thus, the fluorophore's structural integrity is not affected by the pH,as indicated by the similar UV-vis spectra obtained at different pHlevels. Also, the fluorescence emission profile of the fluorophore isnot affected by pH levels, corroborating that the changes are causedafter exposure to ROS.

Factors that Affect Regeneration of Nanoceria Component.

Next, it was determined if nanoceria in the ROS-scavenging chamber ofthe device has the ability to regenerate after exposure to ROS.Utilizing X-ray photoelectron spectroscopy (XPS), we deduced the valencestate of the nanoceria's cerium (Ce⁺³/Ce⁺⁴) is measured at different pHlevels and ROS levels. Proof of regeneration is shown when Ce⁺³ ions arepresent at a concentration level above approximately 55% and Ce⁺⁴ ionsare present in a concentration level greater than approximately 40% ofthe total cerium ions.

FIG. 14 is a chart showing the percentage of each valence state of thecerium element in the polymer-coated nanoceria component at pH 7; pH 7and 1.0 μM H₂O₂; pH 4 and 1.0 μM H₂O₂; pH 7 and 6.0 μM H₂O₂.

XPS analysis indicates that at low ROS concentrations, such as 1 μMH₂O₂, the device's nanoceria has the ability to regenerate. At acidicPh, such as pH 4, or high inflammatory conditions (high ROS), such as 6μM H₂O₂, nanoceria loses its autoregenerative property, leading to adecrease in the device's fluorescence emission, as the DiI-Dex-IOfluorophore is susceptible to ROS attacks.

Multi-Modal DiR-Dex-IO Nanoparticle for a NIR-Fluorescent/MRI MonitoringChamber.

Multimodal monitoring of inflammation through a device, consisting of aROS-scavenging chamber 30 with nanoceria and a multimodal monitoringchamber 40 with DiR-doped dextran-coated iron oxide nanoparticles isshown in FIG. 15. The use of a multimodal DiR-Dex-IO nanoparticle allowsmagnetic resonance imaging (MRI), by the monitoring chamber andrepresents a second embodiment of the present invention.

Multimodal monitoring of inflammation is useful when under elevatedreactive oxygen species (ROS) levels or loss of nanoceria regeneration;the fluorescence emission and the magnetic resonance imaging of thenanocomposite changes and the ROS beacons facilitate tracking with theuse of chemiluminescence, magnetic relaxation or X-ray contrast agents.

In FIG. 15, the ROS scavenging chamber 30 is positioned adjacent tomultimodal monitoring chamber 40 and held in position by an adhesive orglue. In the two chamber device, there is no exchange of particlesbetween the two chambers. Under conditions of no or mild inflammation c,the device's cerium oxide nanoparticle component (nanoceria) in chamber31 scavenges the generated ROS, as ROS production is either nominal ortransient, leading to preservation of the device's fluorescence emissionin chamber 42. However, under conditions of chronic persistentinflammation d, nanoceria in chamber 32 does not have the ability toregenerate quickly enough, thus the excess ROS dim or switch off thedevice's fluorescence emission in chamber 44, due to the fluorophore'ssensitivity to ROS. However, due to the use of iron oxide nanoparticlesin the monitoring chamber 40 and the benefits of magnetic resonanceimaging (MRI), there is an increased MRI signal from the monitoringchamber 44.

Iron oxide nanoparticles are selected because of the wide clinical usein tomographic image acquisitions, and excellent deep tissue resolutionand penetration. DiR is used, which is a near-infrared fluorophore thtcan be used for optical imaging avoiding tissue autofluorescence. Theiron oxide particles were also dextran coated (Dex-IO), as they arewidely used in the clinic as MRI contrast agents. Hence, encapsulatedDiR in Dex-IO nanoparticles (DiR-Dex-IO) are proven to have a dual probefor potentially clinical in vivo inflammation monitoring.

FIG. 16A shows that in the absence of nanoceria, the multimodalDiR-Dex-IO nanoparticles can sense ROS generated by H₂O₂ (1.0 μM), after24-hour exposure to H₂O₂ in pH 7 (physiological pH) via changes influorescence and T2 relaxation times (T2_(in)=165 ms, T2_(fin)=280 ms).FIG. 16B shows that in the presence of nanoceria, the multimodalDiR-Dex-IO nanoparticles can differentiate physiological ROS levelsgenerated by H₂O₂ and the ROS beacons retain their fluorescence emissionintensity with a small change in the T2 relaxation times (T2_(in)=165ms, T2_(fin)=220 ms). Thus, it was determined that a physician using thenanocomposite device will not receive a false positive signal when ROSlevels and pH are physiological.

In the presence of nanoceria, DiR-Dex-IO nanoparticles retainnear-infrared fluorescence and T2 relaxation does not changesignificantly, under exposure to ROS.

In the absence of nanoceria, exposure to ROS leads to a decrease in theDiR-Dex-IO nanoparticle fluorescence where the T2 relaxation increases.This indicates that the device can be used in the clinic forinflammation monitoring with MRI and optical imaging methods. Thechanges in the T2 relaxation times upon exposure to ROS are attributedto nanoparticle clustering.

To elucidate the changes in the T2 relaxation times, Dynamic LightScattering (DLS) was employed. Results indicate that in the absence ofhydrogen peroxide the nanoparticles' average diameter size was 135 nm(FIG. 17A). However, under elevated ROS levels generated by H₂O₂ andwithout nanoceria in the solution to scavenge them, the nanoparticles'average size significantly increased, demonstrating micron-size clusteraggregation with an average particle size of 2860 nm in diameter. (FIG.17B). Hence, this phenomenon justifies the prominent changes in therelaxation times under inflammatory conditions with elevated ROS and nonanoceria. Finally, we assessed the multimodal DiR-Dex-IO nanoparticles'sensitivity towards ROS, observing that even at low concentrations ofH₂O₂ the nanoparticles could induce prominent shifts in the T2relaxation times (FIG. 18) which can be detected by MRI. This indicatesthat these nanoparticles are sensitive ROS imaging probes, thus evenslightly aberrant concentrations of ROS could “turn” the nanoparticles'imaging switch and change the T2 relaxation time.

FIG. 19 is a conceptual diagram of the in vivo inflammation monitoringdevice of the present invention having a single chamber which is asecond embodiment of the device of the present invention. The firstembodiment has two chambers that are not communicating; a first chamberfor the ROS scavenger and a second chamber for monitoring clinicalconditions. The design of the first embodiment is to minimize potentialfluorophore quenching by nanoceria. However, preliminary results with ananocomposite consisting of nanoceria and fluorophore-containingnanoparticles indicated no quenching or photostability issues. Thereforethe second embodiment is a single chamber device containing thereagents, such as nanoceria and DiR-IO-Dex or a nanocomposite.

The reactive oxygen species (ROS) scavenger particles 50 and the imagingagent particles 60 can be interspersed in one chamber. If there are noinflammatory conditions, the nanoceria particles regenerate andfluorescence emission of the imaging agent occurs. In the presence ofpersistent inflammatory conditions, the nanoceria particles do notregenerate and the fluorescence emission of the imaging agent isextinguished.

Dimensions of the device provided by the present invention are givenonly as an illustration, not as a limitation. A macroscale device isapproximately 2 centimeters (cm) in length and approximately 0.4 cm indiameter. A miniature device is approximately 2-4 millimeters (mm) inlength and approximately 1 mm in diameter.

FIG. 20 is a side-view of the fluorescence image of the single-chamberdevice consisting of polymer-coated, polyacrylic acid (PAA) nanoceriaand DiR-Dex-IO nanoparticles after 24-hour-long incubation atphysiological conditions. In FIG. 20, image 1 is the control that showsbright fluorescence intensity with no ROS present, i.e. no H₂O₂ at pH 7.At a physiological H₂O₂ concentration, image 2 also shows brightfluorescence intensity when the device is used in an environment with 1μM H₂O₂ at pH 7. At a non-physiological inflammatory H₂O₂ concentration,image 3 shows a diminished fluorescence intensity in a range ofapproximate 0.9-1.0×10⁵ in an environment with 6 μM H₂O₂ at pH 7, and ata non-physiological chronic inflammatory level H₂O₂ concentration andnon-physiological pH (6 μM H₂O₂, pH 4), image 4 shows a greatlydecreased fluorescence intensity in the range of approximately0.1−0.3×10⁵.

FIG. 21 is a top-view of an MRI image of the single-chamber consistingof polymer-coated, polyacrylic acid (PAA) nanoceria and DiR-Dex-IOnanoparticles after 24-hour-long incubation at physiological conditions.In FIG. 21, Image 5 is of a T2 relaxation time when there are no ROSi.e. no H₂O₂ at pH 7. Image 6 shows T2 relaxation time in the presenceof 1 μM H₂O₂, at pH 7. T2 relaxation times are shown innon-physiological conditions in Image 7 with 6 μM H₂O₂ at pH 7 and inImage 8 with 6 μM H₂O₂ at pH 4 which represents the most severecondition with high ROS and low pH. The T2 relaxation times increase asthe physiological conditions deviate from normal conditions.

In FIGS. 20 and 21, it is demonstrated that a single chamber device ofthe present invention distinguishes between physiological andinflammatory conditions. Overall, in the absence of ROS or at moderateROS levels associated with transient and mild inflammation, thenanoceria in the device is able to scavenge ROS. As a result,fluorescence emission is retained and the T2 relaxation times arecomparable to those observed under normal physiological conditions.

However, at high levels of ROS associated with chronic and acuteinflammation or under non-physiological conditions manifested withdecreases in the pH levels, the fluorescence emission of the devicesignificantly lowers and the T2 relaxation times drastically increase.Thus, under acute conditions, nanoceria cannot scavenge ROS, changingthe device's fluorescence and MRI signatures and signaling therapeuticintervention.

In summary, the present invention provides a therapeutic device which isdesigned to have applications in a broad range of ailments with apro-inflammatory component, such as Crohn's disease, inflammatory boweldisease, ulcerative colitis, cystic fibrosis, sepsis, cardiovasculardisease, arthritis, multiple sclerosis, and Alzheimer's. Furthermore,the use of ROS agents in cancer chemotherapy is reported in severalrepresentative publications, such as, Chung, H. Y., et al., “Molecularinflammation: underpinnings of aging and age-related diseases.” AgeingRes Rev, 2009. 8(1): 18-30 and Shacter, E., et al., in “Oxidative stressinterferes with cancer chemotherapy: inhibition of lymphoma cellapoptosis and phagocytosis” Blood, 2000. 96(1): p. 307-13.

Thus, the two-chamber or single chamber device of the present inventioncan be utilized as an implant in cancer therapeutic regimes. Also,patients with transplants or prosthetic devices may utilize thecerium-oxide-nanoparticle-based device for the in vivo monitoring of thepost-operative inflammatory response.

The device of the present invention can be miniaturized and integratedin a prosthetic organ. Moreover, as the nanoparticles' cavity can host aplethora of probes, the device may be modified in order to be specificto certain ROS species, such as hydroxyl, superoxide, nitroxyl, peroxideand other free radicals, while having multimodality through the presenceof fluorescent, chemiluminescent, magnetic and X-ray agents. Also, thecerium oxide nanoparticles protect the therapeutic device's fluorophoreagainst UV radiation, hence making the device suitable for clinicalapplications, such as chemotherapy, and industry-related activities,including, but not limited to prosthesis, post transplant operations, UVprotection, chemical synthesis, pharmaceutical research and development,basic and clinical research.

While the invention has been described, disclosed, illustrated and shownin various terms of certain embodiments or modifications which it haspresumed in practice, the scope of the invention is not intended to be,nor should it be deemed to be, limited thereby and such othermodifications or embodiments as may be suggested by the teachings hereinare particularly reserved especially as they fall within the breadth andscope of the claims here appended.

We claim:
 1. A nanocomposite system encapsulating nanoparticle elementsnot present in a living body for detection of reactive oxygen species,said nanocomposite system comprising a miniature device in a form of achamber containing therein nanocomposite including a reactive oxygenspecies (ROS)-scavenging nanoparticle component and a reportingnanoparticle component that comprises nanoparticles including one ormore imaging agent, and having an exterior semi-permeable membraneencapsulating both nanoparticle components within the chamber; saidROS-scavenging nanoparticle component comprising polymer coated ceriumoxide nanoparticles, and said nanocomposite comprising a multimodalnanocomposite formed by chemical conjugation of the polymer coatedcerium oxide nanoparticles and the reporting nanoparticle component;said (ROS)-scavenging nanoparticle component and said reportingnanoparticle component communicating with an aqueous environment outsideof said chamber through the semi-permeable membrane that permitsdiffusion of the reactive oxygen species therethrough; saidROS-scavenging nanoparticle component of the nanocomposite protectingthe reporting nanoparticle component from the reactive oxygen speciesunder the normal physiological condition; said reporting nanoparticlecomponent in the chamber incurring an imaging detectable change in thepresence of an elevated non-physiological level of the reactive oxygenspecies associated with an aberrant clinical condition, therebyindicating the presence of an elevated non-physiological level of thereactive oxygen species.
 2. The system of claim 1, wherein saidmultimodal nanocomposite is formed by conjugation of propargylatedpolyacrylic acid coated cerium oxide nanoparticles and azide-carryingindotricarbocyanine polyacrylic acid-coated iron oxide (DiR-PAA-IO). 3.The system of claim 1, wherein said multimodal nanocomposite is formedby carbodiimide conjugation of polyacrylic acid coated cerium oxidenanoparticles and aminated indotricarbocyanine dextran-coated iron oxide(DiR-Dex-IO).
 4. The system of claim 1, wherein the miniature device isapproximately 2 to 4 millimeters (mm) in length and approximately 1 mmin diameter.
 5. The system of claim 1, wherein said polymer-coatedcerium oxide nanoparticles are coated with dextran or polyacrylic acid.6. The system of claim 1, wherein said imaging agent comprises at leastone of a fluorophore capable of fluorescence emission, achemiluminescent agent, a magnetic relaxation agent, and an X-raycontrast agent.
 7. The system of claim 6, wherein said imaging agentcomprises a fluorophore selected from at least one of octodecylindocarbocyanine (DiI), indodicarbocyanine (DiD), indotricarbocyanine(DiR), cyanine 5.5 (Cy5.5), and indocyanine green (ICG).
 8. The systemof claim 6, wherein the reporting nanoparticle component comprisesnanoparticles of at least one of iron oxide, bismuth, europium,gadolinium and chelates thereof.
 9. The system of claim 6, wherein thenanoparticles including one or more imaging agent are coated with atleast one polymer selected from dextran, polyacrylic acid,hyper-branched polyester, poly-lactic-co-glycolic acid, and mixturesthereof.
 10. A nanocomposite system encapsulating nanoparticle elementsnot present in a living body for detection of reactive oxygen species,said nanocomposite system comprising a miniature device in a form of achamber containing therein nanocomposite including polymer-coated ceriumoxide nanoparticles and imaging nanoparticles and having an exteriorsemi-permeable membrane encapsulating the nanoparticles within thechamber; said polymer-coated cerium oxide nanoparticles and said imagingnanoparticles communicating with an aqueous environment outside of saidchamber through the semi-permeable membrane that permits diffusion ofthe reactive oxygen species therethrough; said polymer-coated ceriumoxide nanoparticles of the nanocomposite protecting the imagingnanoparticles from the reactive oxygen species under the normalphysiological condition; said imaging nanoparticles in the chamberincurring an imaging detectable change in the presence of an elevatednon-physiological level of the reactive oxygen species associated withan aberrant clinical condition, thereby indicating the presence of anelevated non-physiological level of the reactive oxygen species.
 11. Thesystem of claim 10, wherein said nanocomposite comprises multimodalnanocomposite formed by conjugation of propargylated polyacrylic acidcoated cerium oxide nanoparticles and azide-carrying indotricarbocyaninepolyacrylic acid-coated iron oxide (DiR-PAA-IO).
 12. The system of claim10, wherein the imaging nanoparticles are nanoparticles of at least oneof iron oxide, bismuth, europium, gadolinium and chelates thereof. 13.The system of claim 10, wherein the imaging nanoparticles are coatedwith at least one polymer selected from dextran, polyacrylic acid,hyper-branched polyester, poly-lactic-co-glycolic acid, and mixturesthereof.
 14. The system of claim 10, wherein the imaging nanoparticlesare polymer-coated nanoparticles of iron oxide for magnetic resonanceimaging detection of the reactive oxygen species, or polymer-coatednanoparticles of iron oxide containing a fluorophore for magneticresonance imaging and fluorescence imaging detections of the reactiveoxygen species.
 15. The system of claim 10, wherein said nanocompositecomprises multimodal nanocomposite formed by carbodiimide conjugation ofpolyacrylic acid coated cerium oxide nanoparticles and aminatedindotricarbocyanine dextran-coated iron oxide (DiR-Dex-IO).
 16. Thesystem of claim 15, wherein the miniature device is approximately 2 to 4millimeters (mm) in length and approximately 1 mm in diameter.
 17. Thesystem of claim 15, wherein the polymer-coated cerium oxidenanoparticles are coated with dextran or polyacrylic acid.
 18. Thesystem of claim 10, wherein the imaging nanoparticles include one ormore imaging agent comprising at least one of a fluorophore capable offluorescence emission, a chemiluminescent agent, a magnetic relaxationagent, and an X-ray contrast agent.
 19. The system of claim 18, whereinthe imaging agent comprises a fluorophore selected from at least one ofoctodecyl indocarbocyanine (DiI), indodicarbocyanine (DiD),indotricarbocyanine (DiR), cyanine 5.5 (Cy5.5), and indocyanine green(ICG).
 20. A nanocomposite system encapsulating nanoparticle elementsnot present in a living body for detection of reactive oxygen species,said nanocomposite system comprising a miniature device in a form of achamber containing therein nanocomposite comprising polymer-coatedcerium oxide nanoparticles and polymer-coated nanoparticles containingone or more imaging agent, and having an exterior semi-permeablemembrane encapsulating the nanoparticles within the chamber; saidpolymer-coated cerium oxide nanoparticles and said imaging nanoparticlescommunicating with an aqueous environment outside of said chamberthrough the semi-permeable membrane that permits diffusion of thereactive oxygen species therethrough; said polymer-coated cerium oxidenanoparticles of the nanocomposite protecting the imaging nanoparticlesfrom the reactive oxygen species under the normal physiologicalcondition; said imaging nanoparticles in the chamber incurring animaging detectable change in the presence of an elevatednon-physiological level of the reactive oxygen species associated withan aberrant clinical condition, thereby indicating the presence of anelevated non-physiological level of the reactive oxygen species.